Foot and mouth disease (FMD) is a devastating and extremely contagious disease of livestock. Its severity is such that the Office International des Epizooties has listed it as an A disease. All species of the order of Artiodactyla, such as, but not limited to, pig, hog, javelina, hippopotamus, camel, llama, mouse deer, giraffe, okapi, deer, pronghorn, antelope, cattle, goat, and sheep, are susceptible to infection with FMD. The financial losses due to infection from the FMD Virus (FMDV) are significant. For example, there are direct losses due to deaths in young animals, loss of milk and loss of meat, as well as decreases in product(s) yield. The costs associated with eradication of infected animals, as well as the costs required to limit the spread of FMDV to non-infected animals, are high. Additionally, there are indirect losses due to the imposition of trade restrictions.
The causative agent of FMD is foot-and-mouth disease virus (FMDV), an aphthovirus of the Picornaviridae family (Bittle et al., 1982 and Fross et al., 1984). The FMDV genome consists of a single RNA positive strand of approximately 8,000 nucleotide bases. The viral RNA is initially translated as a single polypeptide that is subsequently cleaved by viral-encoded proteases to produce four structural capsid proteins (VP1-VP4) and four non-structural proteins (2C, 3A, 3ABC and 3D). The coding region for structural and nonstructural proteins is shown schematically in FIG. 1.
FMDV is antigenically heterogeneous. Seven distinct serotypes have been recognized: O, A, C, ASIA1, SAT1, SAT2 and SAT3 (SAT=Southern African Territories). Each serotype of FMDV is antigenically distinct from the other six serotypes. Serotype A viruses are the most variable, having more than 30 subtypes. Furthermore, each serotype can be subdivided into antigenically distinct multiple subtypes. The serotypes of FMDV were originally identified by cross-immunity experiments in animals. Although an animal that has recovered from infection with one serotype is resistant to challenge by that same serotype, it still remains susceptible to infection by any of the other serotypes.
The different serotypes of FMDV are most prevalent in certain geological areas. For instance, in Asia serotypes A, O, and ASIA1 are most common; in Europe and South America, serotypes A, O, and C are found; and in Africa, serotypes A, O, and SAT are prevalent.
Following infection with FMDV, specific antibodies (IgG and/or IgM, IgA) against the structural proteins (SPs) and non-structural proteins (NSPs) appear. The antibody titers rapidly increase over time and remain high. Thus, the presence of specific FMDV antibody in a sample indicates that the animal from which the sample was collected has had contact with FMDV or an antigen derived from FMDV (such as, but not limited to, animals that have been vaccinated).
Diagnosis of FMD
Primary diagnosis of FMDV commonly involves recognition of typical clinical signs in affected animals. Clinical signs of FMD are essentially similar in all species although the severity may vary considerably. The principal signs are pyrexia, followed by vesicle formation in the mouth and on the feet. Vesicle formation in the mouth causes the animals to salivate. The vesicle formation on the feet causes lameness.
Serological diagnosis of FMD is determined by the presence of FMDV-specific antigens or antibodies in animals suspected of having been infected. The current method of detection is by an enzyme-linked immunosorbent assay (ELISA) or by a virus neutralization test. These methods require multiple steps, are time consuming, and are tedious to perform. Additionally, these assays require long incubation times, resulting in a significant delay in the diagnosis. Time is critical, as FMD is highly contagious. Thus any delay in diagnosis will result in the rapid spread of the disease to uninfected animals, causing a greater loss in product, and subsequently a greater financial loss.
The invention presented herein overcomes these insufficiencies by providing a rapid, one-step method of detecting antibodies and/or antigens in a fluid sample from an animal suspected of having been infected. Additionally, the method presented herein allows for the rapid determination of protection from infection in those animals that have been vaccinated. Furthermore, the disclosed invention is able to determine if an animal has been exposed to FMDV, either by infection or by vaccination, within 30 minutes.
Clinical Symptoms of FMD are not a Reliable Indicator of FMDV Infection
The invention provided herein allows for the rapid detection of antibody to FMDV. Vesicular material is not always available from animals that have been infected with FMDV, thus an additional advantage provided by the present invention is that a liquid sample from animals suspected of having been exposed to FMDV are diagnosed within 30 minutes. Current methodology requires multiple steps (each of which is a potential source of error) and days of incubation(s) prior to obtaining a result. Additionally, diagnosis of FMD by clinical signs alone is difficult, especially for sheep and goats, in which clinical signs are often mild (Barnett, P. V et al., 1999 and Callens, M., K. et al., 1998). Furthermore, several other vesicular virus infections, including, but not limited to, those caused by swine vesicular disease (SVD) virus and vesicular stomatitis virus (VSV), cannot be distinguished from FMDV infection by the clinical findings. Thus, a rapid diagnosis of the infectious agent is critical.
Moreover, FMDV can establish a persistent or carrier stage in ruminants in the absence of any clinical manifestation of the disease. Such carrier animals are a source of new outbreaks of the disease. Therefore, there is a long sought, yet unfulfilled, need for a rapid serological method in which infected and/or asymptomatic carrier animals are identified. Additionally, there is a long sought, yet unfulfilled, need for an assay system that will distinguish between animals that are protected from FMDV infection, i.e., those that have been vaccinated, from animals that have been infected with FMDV. Since both vaccination against FMDV and FMDV infection induced antibodies to the structural capsid proteins, assays that detect the structural capsid protein alone are insufficient to differentiate vaccinated animals from those that have been infected. Thus, antibodies against structural proteins can only be used in vaccine-free regions, such as, but not limited to, the USA or the UK. Assay systems that can only detect these anti-structural protein antibodies are therefore not useful in regions where animals are vaccinated. Since unvaccinated animals present in the geographic regions where vaccinations are given remain susceptible to infection with FMDV, there is a long sought, yet unfulfilled, need for a diagnostic test that will differentiate an infected animal from one that has been vaccinated (and thus protected from infection). The invention disclosed herein provides such an assay system.
The present invention further provides an assay system that will measure the efficacy of vaccinations. The present invention disclosed herein provides for the current deficiencies in the art by providing an assay system that allows for the rapid detection of antibodies to both SPs and NSPs, thereby allowing for vaccinated animals to be differentiated from those that are infectious. Finally, the present invention provides a detection method that allows for rapid analysis of the geographic distribution of specific FMDV serotypes and/or serotype subtypes for epidemiological studies.
Thus the present invention provides a rapid immunochromatographic assay wherein E. coli-expressed recombinant FMDV structural and non-structural proteins are incorporated into a solid support for the capture of antibodies to FMDV in animal body fluids. The immunochromatographic assay disclosed herein allows for the diagnosis of FMDV infection, for the identification of FMDV carriers, as well as for the ability to differentiate between an infected animal and an animal that has been vaccinated (and thus protected from FMDV).